nh 4 cl  (MedChemExpress)

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: a Representative images (left) and quantitative analysis (right) of CMA flux, indicated by the number of KFERQ puncta, in GSC and non-GSC cells stably expressing PAmCherry-KFERQ or PAmCherry-KFSDA (a mutant KFERQ sequence that cannot be recognized by HSC70). n = 40 randomly selected cells. Scale bar, 10 μm. b In vitro binding and uptake assay of GAPDH by the lysosome in GSC and non-GSC cells. n = 3 independent experiments. c Immunoblotting (IB) analyses for indicated proteins after treating with 10 μM MG132, 5 mM 3-MA, or LN (10 μM leupeptin and 20 mM NH 4 Cl) in GSC cells (M83 and 23) and non-GSC cells (LN229 and U251), respectively. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin. n = 3 independent experiments. d Representative images (left) and quantification (right) of CMA activity detection by KFERQ puncta numbers in GSCs (M83 and 456) and their corresponding DGCs. n = 40 randomly selected cells. Scale bar, 10 μm. e IB analyses for LAMP2A and CMA-associated chaperone proteins (HSC70, HSP40 and HSP90) in lysosomes isolated from the indicated GSCs and DGCs, with LAMP1 serving as a lysosomal marker. f Sphere-forming frequency of GSC M83 and 456 cells with indicated modifications. sh-C, control for knock down. Vec, a control vector. WT, wide-type. Representative H&E images of mouse brain sections ( g ) and quantitative analysis of tumor volume ( h ) from athymic (BALB/c Nude) mice intracranially implanted with the GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. i Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 4 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test ( a, b, d ), two-sided likelihood ratio test ( f ), one-way ANOVA with Dunnett’s multiple comparisons test ( h ) or log-rank test ( i ). ns, not significant. Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Stable Transfection, Expressing, Mutagenesis, Sequencing, In Vitro, Binding Assay, Western Blot, Activity Assay, Isolation, Marker, Control, Knockdown, Plasmid Preparation

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: a, b Gene set enrichment analysis highlighted the main biological processes significantly altered due to LAMP2A KD in GSCs, derived from RNA-seq data ( GSE181556 , a ) and proteomic data (PXD027069, b ). These pathways of interest especially immune-related pathways are indicated in red. c IB analyses for indicated proteins in GSC M83 cells expressing sh-C, sh-MST4 or sh-LAMP2A. d, e IP-IB analyses for indicated proteins in HEK293T cells transduced with the indicated plasmids. f IB for TSC1 (top) and TSC2 (bottom) in HEK293T cells subjected to indicated modifications, subsequently treated with CHX (100 μg/mL) for the indicated time. g Quantification of TSC1 (left) and TSC2 (right) protein levels in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. h, i IB analyses for TSC1/2 protein levels in WCL and lysosomal fractions of GSC M83 cells, with or without LAMP2A KD ( h ) or HSC70 KD ( i ). j IB analyses for TSC1 and TSC2 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH 4 Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. k Representative H&E images of mouse brain sections (left) and quantification of tumor volume (right) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. l Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 5 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-sided Fisher’s exact test ( a, b ), two-way ANOVA with Bonferroni’s multiple comparisons test ( g ), one-way ANOVA with Dunnett’s multiple comparisons test ( k ) or log-rank test ( l ). ns, not significant. Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Derivative Assay, RNA Sequencing, Expressing, Transduction

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: GSEA enrichment plots for the cGAS/STING signaling pathway ( a ) and the interferon α/β signaling cascade ( b ) in GSCs lacking LAMP2A. c, d Impact of IR (6 Gy) on the activation of STING/TBK1 in GSC M83 cells, both with and without LAMP2A KD ( c ) or MST4 KD ( d ). e, f Quantitative real-time PCR (qRT-PCR) analysis of mRNA levels of CGAS ( e ) and STING ( f ) in GSC M83 cells transduced with sh-C, sh-MST4 or sh-LAMP2A. n = 3 independent experiments. Analysis of the relationship between mRNA levels and methylation status of CGAS ( g ) and STING ( h ) in GBM samples derived from TCGA datasets. i IB analyses for cGAS and STING in HEK293T cells transfected with Vec, TET1, TET2 or TET3. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin. j IB analyses for indicated proteins in HEK293T cells with indicated modifications. k IP-IB for indicated proteins in HEK293T cells overexpressing Myc-TET3 wild-type (WT) or mutant variant (Mut). l IB analysis for TET3 in HEK293T cells overexpressing Myc-TET3-WT or -Mut, following treatment with CHX (100 μg/mL) for indicated time. m Quantification of TET3 protein level in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. IB analyses for indicated proteins in WCL and lysosomal fractions of GSC M83 cells with or without LAMP2A KD ( n ) or HSC70 KD ( o ). p IB analysis for TET3 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH 4 Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using two-sided Kolmogorov-Smirnov test ( a, b ), one-way ANOVA with Dunnett’s multiple comparisons test ( e, f ), two-sided Pearson correlation test ( g, h ) or two-way ANOVA with Bonferroni’s multiple comparisons test ( m ). Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction, Methylation, Derivative Assay, Transfection, Mutagenesis, Variant Assay

Journal: Cancer Communications

Article Title: m 6 A‐modified EHD1 controls PD‐L1 endosomal trafficking to modulate immune evasion and immunotherapy responses in lung adenocarcinoma

doi: 10.1002/cac2.70052

Figure Lengend Snippet: EHD1 promoted the endocytic recycling of PD‐L1 and inhibited its lysosomal degradation. (A) Colocalization analysis of PD‐L1 with RAB11 and LAMP1 in shCtrl and sh EHD1 LUAD cells. (B) The relative PD‐L1 expression in LUAD cell surface regulated by EHD1 was detected by FCM. Representative FCM histograms (left), and quantification data (right) are displayed. (C‐D) The surface level of remaining PD‐L1 after EHD1 knockdown was detected by (C) FCM analysis and (D) IP experiments at the specified time points. (E) A CHX chase assay was performed to analyze the stability of the PD‐L1 protein in shCtrl and sh EHD1 LUAD cells. The cells were treated with CHX (10 µg/mL) for the designated time. (F‐G) Western blotting analysis was performed to measure PD‐L1 expression in shCtrl and sh EHD1 LUAD cells treated with the proteasome inhibitor MG132 (F) or the lysosomal inhibitor NH 4 Cl (G) for 8 h. (H) Ubiquitination IP assays validated the degradation of PD‐L1 in LUAD cells transfected with the total‐Ub, UbK63 or UbK48 plasmid. NH 4 Cl was used for treatment before cell lysis. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. CHX, Cycloheximide; DAPI, 4',6‐diamidino‐2‐phenylindole; EHD, Eps15 homology domain; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; HA‐Ub, Hemagglutinin ubiquitin; IP, Immunoprecipitation; LAMP1, Lysosome‐associated membrane protein 1; LUAD, Lung adenocarcinoma; PD‐L1, Programmed cell death 1 ligand 1; PE, Phycoerythrin; RAB11, Ras‐Related Protein 11; shCtrl, short hairpin RNA control; shEHD1, short hairpin RNA for EHD1; Total‐Ub, Total ubiquitin; UbK48, Ubiquitination at the lysine 48 residue; UbK63, Ubiquitination at the lysine 63 residue; FCM, Flow cytometry.

Article Snippet: For the cycloheximide (CHX) chase assay, cells were treated with 10 μg/mL CHX (HY‐12320, MedChemExpress) for 0, 1, 2, 3, and 4 h. For the protein degradation pathway assay, cells were treated with the proteasome inhibitor MG132 (10 μmol/L, HY‐13259, MedChemExpress) or lysosomal inhibitor NH 4 Cl (20 mmol/L, 12125‐02‐9, Aladdin) for 8 h. Then cells were collected and lysed on ice, subsequently verified by Western blotting.

Techniques: Expressing, Knockdown, Western Blot, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Lysis, Two Tailed Test, Immunoprecipitation, Membrane, shRNA, Control, Residue, Flow Cytometry

Journal: Cancer Communications

Article Title: m 6 A‐modified EHD1 controls PD‐L1 endosomal trafficking to modulate immune evasion and immunotherapy responses in lung adenocarcinoma

doi: 10.1002/cac2.70052

Figure Lengend Snippet: YTHDF1 regulated EHD1 expression and its mRNA stability. (A‐B) The typical m 6 A motif RRACH in EHD1 mRNA was identified using SRAMP (A) and MEME (B) (R = A or G; H = A, U or C). (C) Enrichment of m 6 A modifications on EHD1 mRNA was detected by MeRIP‐qPCR. (D) Venn diagram showing the regulators of m 6 A transcriptional modifications upstream of EHD1 , which were predicted by RM2target, RMBase, and RMVAR. (E) The binding of EHD1 mRNA to YTHDF1 or IGF2BP1 was predicted using RPISeq. RF and SVM classifiers are used to predict RNA‐protein interaction probabilities based on sequence features; values above 0.5 suggest a potential interaction. (F) The correlation between the m 6 A regulators YTHDF1 and EHD1 was characterized by Spearman correlation analysis based on mRNA expression levels using the TCGA‐LUAD dataset. (G‐H) EHD1 mRNA and protein levels in LUAD cells with YTHDF1 knockdown were measured by qRT‐PCR and Western blotting. (I‐J) An Act D pulse‐chase experiment was performed to determine the stability of EHD1 mRNA in the indicated cells treated with 5 µg/mL Act D. (K‐L) EHD1 expression in shCtrl and sh YTHDF1 LUAD cells treated with (K) MG132 or (L) NH 4 Cl for 8 h was detected by Western blotting analysis. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Act D, Actinomycin D; EHD1, Eps15 homology domain 1; IGF2BP1, Insulin like growth factor 2 MRNA binding protein 1; IgG, Immunoglobulin G; LUAD, Lung adenocarcinoma; m 6 A, N6‐methyladenosine; MeRIP‐qPCR, Methylated RNA immunoprecipitation‐quantitative polymerase chain reaction; RF, Random forest algorithm; shCtrl, short hairpin RNA control; shYTHDF1, short hairpin RNA for YTHDF1; SVM, Support vector machine; YTHDF1, YTH N6‐Methyladenosine RNA Binding Protein F1; SD, Standard deviation.

Article Snippet: For the cycloheximide (CHX) chase assay, cells were treated with 10 μg/mL CHX (HY‐12320, MedChemExpress) for 0, 1, 2, 3, and 4 h. For the protein degradation pathway assay, cells were treated with the proteasome inhibitor MG132 (10 μmol/L, HY‐13259, MedChemExpress) or lysosomal inhibitor NH 4 Cl (20 mmol/L, 12125‐02‐9, Aladdin) for 8 h. Then cells were collected and lysed on ice, subsequently verified by Western blotting.

Techniques: Expressing, Binding Assay, Sequencing, Knockdown, Quantitative RT-PCR, Western Blot, Pulse Chase, Two Tailed Test, Methylation, RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA, Control, Plasmid Preparation, RNA Binding Assay, Standard Deviation